The broad aim of this proposal is to understand how the head of bacteriophage T4 is assembled and filled with DNA. We will concentrate on one aspect of this process - namely, the proteolytic cleavage reactions that accompany T4 assembly. This aspect has been chosen for study because of the ubiquitous occurrence of cleavage during the assembly of a variety of phage and animal viruses. By assaying for a specific small fragment cleaved from a T4 protein (one of the internal peptides) we have recently been able to demonstrate cleavage in vitro. In vitro cleavage has been found to require specifically the product of T4 gene 21. This proteolytic factor can be detected in cells infected by T4 mutants blocked in an early stage of head formation but cannot be detected in T4 wild-type infected cells. We will use the in vitro assay for cleavage to further study the process. Our aims are: (1) to identify the precursors of the T4 internal peptides, (2) to determine the mechanism and specificity of cleavage, (3) to determine the fate of the T4 proteolytic factor when head assembly goes to completion, (4) to purify and characterize the T4 proteolytic factor, (5) to study the possible involvement of host factors in the cleavage process, (6) to genetically modify the head assembly pathway of T4, (7) to isolate additional T4 mutants with a modified gene 21, and (8) to test a proposed model to explain the role of cleavage in assembly.